Recombinant tPA remains the only FDA approved and the most beneficial proven intervention for treatment of ischemic stroke. However, clinical use of tPA is limited due to its narrow therapeutic window and increased risk of hemorrhagic transformation (HT). In this proposal, we hypothesize that therapeutic inhibition of PI3Kg with selective PI3K? inhibitors, alone or in combination with tPA, protects against the ischemia- and tPA-induced brain injury and HT and increases therapeutic window for tPA treatment of ischemic stroke. The molecular basis for our idea is that genetic deletion of PI3Kg not only reduces stroke-induced BBB disruption and tissue infarction but also almost completely blocked cerebral hemorrhage induced by tPA administered at 6 hours after stroke. Our data are promising and suggest a critical role of PI3K? in tPA-induced HT and brain injury after ischemic stroke. Further, our pilot data show that inhibition of PI3Kg with the PI3K? inhibitor AS605240 significantly protects against brain injury and reduces fibrin deposition/thrombosis in the ischemic brain after embolic stroke in rats. These exciting preliminary data suggest that PI3Kg may represent a promising therapeutic target for treating acute ischemic stroke. In this application, we propose to develop a new stroke therapy by therapeutic targeting of PI3Kg with small molecule selective PI3K? inhibitors (AS605240, AS252424) for treatment of acute ischemic stroke and to investigate molecular mechanisms underlying the PI3K? inhibition-mediated beneficial effects. We now propose three aims to investigate this new stroke therapy. The experiments will be performed on normotensive and hypertensive rats in a clinically relevant rat model of thromboembolic stroke. In Aim 1, we will determine the effects of AS605240 (and AS252424), alone and in combination with tPA, treated at 6 hrs after focal embolic stroke in rats. We will identify the optimal dosage and the extended therapeutic time window for the PI3K? inhibition alone and in combination with tPA for treatment of acute stroke. Brain infarction, neurological deficits, brain hemorrhage, and animal mortality will be examined 72h after stroke. In Aim 2, we will define the mechanisms that underlie the beneficial effects of the PI3K? inhibition alone or in combination with tPA in the treatment of acute stroke. BBB damage will be assessed by the spatial and tem poral analysis of key BBB proteins and potential correlation with changes in activation/expression of NF-?B, CD147, MMP-9 and/or MMP-3 on cerebral vessels. Thrombus formation in the ischemic brain after stroke will be assessed by analyzing fibrin deposition within and around the cerebral vessel wall and the expression of prothrombotic factors (PAI-1, tissue factor-TF) in the ischemic brain. In Aim 3, we will assess long term outcomes of the PI3K ? inhibition by AS605240 alone and in combination with tPA treated at 6 hrs after embolic stroke. Animals will be followed over 5 weeks with a battery of behavioral tests. At the end, brains are removed for measurements of lesion size, fibrin deposition/thrombosis, cerebral vascular density/potency, and their correlation with the expression of proangiogenic factors (VEGF, Ang-1) and prothrombotic factors (PAI-1, tissue factor-TF). If successful, these proposed experiments should provide new insights of how the class IB PI3Kg signaling contributes to the ischemia- and tPA-induced HT and brain injury and may ultimately yield new therapies to treat acute ischemic stroke and to minimize the adverse effect of tPA on ischemic neurovascular damage, leading to improved long term outcomes after ischemic stroke.